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Construction of two different flow cell systems. (A) Overview of the flow cell system constructed in this study. (B) Tubular observation cell for the quantification of cell adhesion. (C) Rectangular observation cell for the observation of cell behavior in a liquid flow.
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It is important to characterize how medically, industrially, or environmentally important bacteria adhere to surfaces in liquid flows in order to control their cell adhesion and subsequent biofilm formation. Acinetobacter sp. Tol 5 is a remarkably sticky bacterium that autoagglutinates through the adhesive nanofiber protein AtaA, which is applicabl...
Citations
... A rectangular flow chamber system, which we have previously reported (Furuichi et al., 2018), was used with slight modifications: a syringe pump (Legato 200; KD Scientific, Holliston, MA) was directly connected to a square glass tube 50 mm in length and 1 mm in every internal dimension without using a three-way stopcock valve. Tol 5 cells were suspended in BS-N buffer at an OD 660 of 0.2, and the suspension was subjected to sonication to break up the cell clumps. ...
... Live images of the behavior of Tol 5 cells during their adhesion and detachment to/from the inner surface of the glass tube were recorded under a digital microscope (VHX-200; Keyence, Osaka, Japan). Wall shear stress (τ) was calculated using Eq. 1, as previously described (Furuichi et al., 2018): ...
... To investigate the behavior of Tol 5 cells on the MPC polymer surface in the presence of shear forces, we observed Tol 5 cells on the surface by using a flow chamber system with a square glass tube ( Figure 5A) (Furuichi et al., 2018). The glass tube with or without the MPC polymer coating was filled with a Tol 5 cell suspension and incubated for 10 min. ...
Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.
... Considering the quite short contact time (<100 ms) of a cell surface with an AFM probe, a single Tol 5 cell can instantaneously attain strong adhesion to a silicon probe. Recently, we showed that autoagglutination through AtaA significantly contributes to biofilm formation of Tol 5 cells by triggering the formation of the biofilm [36,37]. However, the current result demonstrated that a single Tol 5 cell itself has the ability to very strongly adhere to a surface through the quick interaction of AtaA with the surface. ...
The sticky bacterium Acinetobacter sp. Tol 5 adheres to various material surfaces via its cell surface nanofiber protein, AtaA. This adhesiveness has only been evaluated based on the amount of cells adhering to a surface. In this study, the adhesion force mapping of a single Tol 5 cell in liquid using the quantitative imaging mode of atomic force microscopy (AFM) revealed that the adhesion of Tol 5 was near 2 nN, which was 1-2 orders of magnitude higher than that of other adhesive bacteria. The adhesion force of a cell became stronger with the increase in AtaA molecules present on the cell surface. Many fibers of peritrichate AtaA molecules simultaneously interact with a surface, strongly attaching the cell to the surface. The adhesion force of a Tol 5 cell was drastically reduced in the presence of 1% casamino acids but not in deionized water (DW), although both liquids decrease the adhesiveness of Tol 5 cells, suggesting that DW and casamino acids inhibit the cell approaching step and the subsequent direct interaction step of AtaA with surfaces, respectively. Heterologous production of AtaA provided non-adhesive Acinetobacter baylyi ADP1 cells with a strong adhesion force to AFM tip surfaces of silicon and gold.
... In instances where aggregates -and even biofilms-detach or float, a relationship between autoaggregation and biofilm mass may not be seen in conventional laboratory assays that measure biofilms on horizontal solid supports but may be more visible on vertically mounted surfaces [35]. Biofilms on horizontal surfaces benefit from gravitational pull on planktonic cells and biofilm aggregates while autoaggregation is less critical to shear stress-mediated biofilm formation in continuous flow systems [55]. In static culture, autoaggregation and biofilm of V. parvula SKV38 formation correlate but in dynamic flow, a non-aggregating Veillonella trimeric autotransporter gene mutant formed six times more biofilm than the wildtype. ...
Autoaggregation, adherence between identical bacterial cells, is important for colonization, kin and kind recognition, and survival of bacteria. It is directly mediated by specific interactions between proteins or organelles on the surfaces of interacting cells or indirectly by the presence of secreted macromolecules such as eDNA and exopolysaccharides. Some autoaggregation effectors are self-associating and present interesting paradigms for protein interaction. Autoaggregation can be beneficial or deleterious at specific times and niches. It is, therefore, typically regulated through transcriptional or post-transcriptional mechanisms or epigenetically by phase variation. Autoaggregation can contribute to bacterial adherence, biofilm formation or other higher-level functions. However, autoaggregation is only required for these phenotypes in some bacteria. Thus, autoaggregation should be detected, studied and measured independently using both qualitative and quantitative in vitro and ex vivo methods. If better understood, autoaggregation holds the potential for the discovery of new therapeutic targets that could be cost-effectively exploited.
... 137 Glass tubular and rectangular cross sectioned ow has been used to study the adhesion of Acinetobacter spp. on PMMA surfaces. 138 In this study, no signicant effect of shear stress level on bacterial adhesion was observed. However, a temporal study of bacterial adhesion has shown an increase in number of bacterial cells on the surface by means of new adhesions to the surface and stacking of bacteria on existing clumps. ...
... Bacterial adhesion tests performed with substrates held upright and upside-down under ow conditions had comparatively lower adhesion on upsidedown surfaces. 138,[142][143][144] Similarly, under non-owing condition, bacterial adhesion on upside-down substrate has been lower than that of upright substrate. 26 Li et al. studied the adhesion of S. aureus on glass slides held at top and bottom in a PPFC under laminar ow and observed adhesion on bottom surface increased over the time, while that on top surface reached a maximum soon aer start of the ow. ...
... Several studies have shown the effect of gravity that relates to lower adhesion of bacteria on the top surface of a ow-cell than on the bottom surface as discussed previously. 138,142 S. aureus tested on titanium substrate with nano-pillars and nano-pores under ow with shear rate of 6 s À1 (z0.006 Pa) resulted in low BE of approximately 15% and 23% respectively. ...
Micro/nanostructured surfaces (MNSS) have shown the ability to inactivate bacterial cells by physical means. An enormous amount of research has been conducted in this area over the past decade. Here, we review the various surface factors that affect the bactericidal efficiency. For example, surface hydrophobicity of the substrate has been accepted to be influential on the bactericidal effect of the surface, but a review of the literature suggests that the influence of hydrophobicity differs with the bacterial species. Also, various bacterial viability quantification methods on MNSS are critically reviewed for their suitability for the purpose, and limitations of currently used protocols are discussed. Presently used static bacterial viability assays do not represent the conditions of which those surfaces could be applied. Such application conditions do have overlaying fluid flow, and bacterial behaviours are drastically different under flow conditions compared to under static conditions. Hence, it is proposed that the bactericidal effect should be assessed under relevant fluid flow conditions with factors such as shear stress and flowrate given due significance. This review will provide a range of opportunities for future research in design and engineering of micro/nanostructured surfaces with varying experimental conditions.
... Similar catch bondtype behavior has been observed for several bacterial adhesins before (Dufrêne & Viljoen, 2020;Hospenthal & Waksman, 2019) and also the TAA AtaA from Acinetobacter sp. Tol 5 mediates stronger binding to glass surfaces and more aggregation under moderate shear stress (Furuichi, Iwasaki, & Hori, 2018). ...
Trimeric autotransporter adhesins (TAAs) comprise a group of virulence‐related proteins in Gram‐negative bacteria. Members of this family bind to extracellular matrix components such as collagen and fibronectin, but also they exhibit several other functions, such as conferring serum resistance and autoaggregation. Autoaggregation promoted by TAAs is homotypic and mediated by the sticky, globular head domains of these lollipop‐like molecules. However, whether TAAs mediate heterotypic interactions (i.e., coaggregation) has not been studied. To address this question, we investigated the coaggregation of two model TAA groups: YadA from the enteropathogenic Yersiniae and the immunoglobulin‐binding Eib proteins from Escherichia coli. To study TAA coaggregation, we coexpressed a fluorescent label together with a particular TAA and followed the aggregative interactions using fluorescence microscopy and quantified the interactions using a novel script implemented in Fiji. Our results show that there is coaggregation between some populations expressing different TAAs, which can be explained by relatively high sequence similarity between the interacting TAAs. Generally, the level of coaggregation correlated with the sequence similarity. However, some TAAs did not interact despite high sequence similarity, showing exclusion of bacteria producing a noncompatible TAA. These data demonstrate that TAAs can mediate bacterial coaggregation, but in some cases prevent coaggregation of bacteria with disparate TAAs. Our results have implications for the ecology of TAA‐producing bacteria, where coaggregation may promote co‐operation whereas exclusion might be an indication of competition.
AtaA, the sticky, long, and peritrichate nanofiber protein from Acinetobacter sp. Tol 5, mediates autoagglutination and is highly adhesive to various material surfaces, resulting in a biofilm. Although the production of the adhesive nanofiber protein is likely to require a large amount of energy and material sources, the relationship between AtaA fiber production and cell growth remains unknown. Here, we report the growth phase-dependent AtaA fiber production in Tol 5. We examined the ataA gene expression in different growth phases using a reporter gene assay with an originally developed reporter plasmid and using reverse transcription-quantitative polymerase chain reaction. Bacterial cells with surface-displayed AtaA at different growth phases were immunostained and analyzed using fluorescence flow cytometry and confocal laser scanning microscopy. The results indicate that Tol 5 modulated the amount of surface-displayed AtaA at the transcriptional level. AtaA production was low in the early growth phase but remarkably increased in the late growth phase, covering the whole bacterial cell with AtaA fibers in the stationary phase. Tol 5 displayed AtaA fibers poorly in the early growth phase and showed less autoagglutination and adhesiveness than those in the stationary phase. Although Tol 5 grew as fast as its ataA-deficient mutant in the early growth phase, the optical density of Tol 5 culture was slightly lower than that of the ataA-deficient mutant in the late growth phase. Based on these experimental results, we propose the growth-phase-dependent production of AtaA fiber for efficient and fast cell growth.
Biofilms are colonies of microorganisms that adhere to a surface, often in a flowing system. They convey advantages to the community of cells within, such as access to nutrients, protection from physicochemical stress, intercellular communication and horizontal gene transfer. In many instances, biofilms are viewed as problematic, for example, causing difficult-to-treat infections or corroding metal surfaces. However, the properties that make biofilms a challenge in some circumstances are beneficial in others. As biofilms have mechanical stability, are resistant to the toxic effects of xenobiotics and are stable for extended time periods, they have potential applications in the continuous production of chemicals and the biodegradation of pollutants. In this chapter, we will present examples of the research that has been conducted on examining single-species biofilms of bacteria and fungi in the biosynthesis of commodity and fine chemicals and the biodegradation of xenobiotics. The merits and disadvantages of biofilms in different applications are discussed.
An enrichment culture strategy was used to obtain natural consortia from soil in order to study the biodegradability of poly(ether-urethanes) (PUE). PUE were synthesized with different aliphatic diisocyanate group, termed PUIPDI and PUHMDI. In addition, PUE/acrylic hybrids, termed PUH90:DEA10 and PUH70:DEA30 consisted of PUHMDI and increasing ratio of 2-(diethylamino)ethyl methacrylate (DEA) were tested. PUIPDI was biodeteriorated through urea-bond hydrolysis by a consortium dominated by Acinetobacter. A higher biodeterioration rate was demonstrated for PUHMDI, which exhibited large surface holes and a greater weight loss. The hydrolysis of ester-urethane bonds and the oxidation of soft-segment ether bonds were the key reactions identified, possibly by Acinetobacter, Mycobacterium, Sphingopyxis and Pseudomonas, which were present in this consortium. Diverse metabolic functions were also predicted. The addition of DEA favored the biodegradability of PUH:DEA hybrids demonstrated by the increased weight loss, swelling degree and the hydrolysis of DEA-ester bond. Urethane and ether bonds degradation were also detected. PUH:DEA hybrids selected even bacterial consortia, most likely by allowing better access to the nutrients. Pseudomonadaceae and Bradyrhizobiaceae families got relevance in these consortia and their outstanding features were cell mobility and quorum sensing. This work has provided a deeper insight into PUE biodegradation by proposing the chemical mechanisms, bacterial taxa and functions that could be implicated.
In this study, we elucidated the formation process of an unconventional biofilm formed by a bacterium autoagglutinating through sticky, long, and peritrichate nanofibers. Understanding the mechanisms of biofilm formation is essential to control microbial behavior and improve environmental biotechnologies. Acinetobacter sp. Tol 5 autoagglutinate through the interaction of the long, peritrichate nanofiber protein AtaA, a trimeric autotransporter adhesin. Using AtaA, without cell growth or extracellular polymeric substances production, Tol 5 cells quickly form an unconventional biofilm. The process forming this unconventional biofilm started with cell–cell interactions, proceeded to cell clumping, and led to the formation of large cell aggregates. The cell–cell interaction was described by DLVO theory based on a new concept, which considers two independent interactions between two cell bodies and between two AtaA fiber tips forming a discontinuous surface. If cell bodies cannot collide owing to an energy barrier at low ionic strengths but approach within the interactive distance of AtaA fibers, cells can agglutinate through their contact. Cell clumping proceeds following the cluster–cluster aggregation model, and an unconventional biofilm containing void spaces and a fractal nature develops. Understanding its formation process would extend the utilization of various types of biofilms, enhancing environmental biotechnologies.
Acinetobacter sp. Tol 5 exhibits an autoagglutinating nature and high adhesiveness to various abiotic surfaces through its bacterionanofiber protein AtaA. We have developed new bacterial immobilization methods utilizing the high adhesiveness of AtaA. We previously reported that salt is essential for the adhesiveness of AtaA. In the current study, we unexpectedly found that Tol 5 cells were not immobilized onto polyurethane foam support during growth in LB medium although AtaA was properly expressed and displayed onto the cell surface. The adhesion of Tol 5 resting cells was not affected by sugars but drastically inhibited by yeast extract and casein hydrolysates such as tryptone and casamino acids technical grade (CA-T). Some amino acids, which are major components of CA-T, partially inhibited the adhesion of Tol 5 cells. Experimental results suggested that oligopeptides might effectively inhibit the cell adhesion. Immobilized cells onto the support through AtaA were detached in CA-T solution. Also, the detached cells could be re-immobilized onto the support without impairing of their adhesiveness by replacing CA-T solution to a basal salt medium. Microscopic observation revealed that breaking of AtaA-mediated cell-cell interaction is important for the detachment of Tol 5 cells from the support. CA-T also inhibited AtaA-mediated autoagglutination and dispersed cell clumps through AtaA. This is the first report on adhesion inhibitors against AtaA and suggests that casein hydrolysates like CA-T would be a powerful tool for controlling AtaA-mediated bacterial immobilization.
